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# Cells counting problem.

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### #1 Ika_WUM

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Posted 16 July 2014 - 01:27 AM

Hi, guys. I need your help.

I’m culturing H9c2 myoblast, for about month now, which should be passaged at 80% confluency. Till now I’ve been using LUNA cell counting device. But I’m suspicious about received results. According to LUNA, the number of cells harvested from bottles are usually a few thousand (5-8). Funny thing is that results do not change that much when I use T-75 instead of T-25.

So lately I've started to count my cells with use of Thoma chamber. And now I’m getting results around 20x10^6 from T-25 flask. Is that even possible? I’ve got Thoma chamber with 0.2mm x 0.2mm dimension so I multiply the average number of cell from medium squares by dilution factor and 250 000.

Could you tell me how many cells are on average on T-25 flask? Where do I make mistake?

Yours Faithfully

### #2 SusieQ

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Posted 16 July 2014 - 04:17 AM

Are the cell numbers you're giving the total amount of cells, or per ml?

The difference with the cells obtained from a T75 and a T25 is that, after trypsinizing, you resuspend your cells in culture medium. The volume of culture medium is different between these flasks; I usually resuspend my cells in 10 ml medium in a T75, whereas I would use 5 ml in a T25. The amount of cells you harvest from the T25 is in a smaller volume than those obtained from the T75, which can result in a equal cell concentration, depending on the amount of cells and the resuspension volume.

The amount of cells you get from either flasks can vary greatly; that depends on the size of the cells and their behaviour. Some cell lines grow in monolayers and some cell lines don't. Also, whether they're adherent or grow in suspension matters.

### #3 Ika_WUM

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Posted 16 July 2014 - 11:28 PM

Right. So, the cell numbers i gave was per ml. And actually i count my cells after centrifuging, always resuspending them in the same amount of medium. I took into account the dilution factor.

If it goes to cells they are adherent and grow in monolayer. Average size of the sells after tripsinization is around 20um.